Gene/environment interactions in the development of various neurological diseases have been well documented. DNA damage, including single-strand breaks (SSBs), is the outcome of one such interaction. Defects in DNA SSB repair (SSBR) may have striking human health consequences. Several neurological diseases have already been identified and characterized that are due to the lack of DNA end-processing activities, catalyzed by enzymes such as aprataxin and TDP1. Human polynucleotide kinase 3'-phosphatase (PNKP) is another SSBR enzyme that processes 3'-P and 5'-OH ends, generated both endogenously and by exogenous genotoxic agents. These DNA termini need to be processed to restore genomic integrity, because unrepaired SSBs would block transcription, which is detrimental in all cells. We hypothesize that SSBs in the transcribed strand of active genes are preferentially repaired via a subpathway of SSBR, which we call transcription-coupled SSBR (TC-SSBR), and that PNKP plays a vital role for 3'-P and 5'-OH end- processing in the transcribed sequences. We have now found that PNKP is present in the mitochondria. The association of PNKP with nuclear and mt RNA polymerases, and preferential association of PNKP with transcribed genes, further supports our hypothesis of preferential repair of actively transcribed genes. Our surprising observation of the association of PNKP with Ataxin-3 (ATXN3), a protein responsible for spinocerebellar ataxia type 3, also called Machado-Joseph Disease (MJD/SCA3), prompted us to investigate PNKP's role in the pathogenesis of the disease. MJD/SCA3 is a fatal, autosomal dominant disorder caused by CAG repeat (poly-Q) expansion in the coding region of the ATXN3 gene. There is no therapy available for this disease. A common pathological feature of poly-Q diseases is the accumulation of intranuclear inclusions. However, the mechanism by which pathogenic ATXN3 (ATXN3-Q72) causes neurodegeneration is still not clearly understood. Our preliminary data showed that the pathological form of ATXN3 blocked PNKP-mediated SSBR activities in vitro. It is thus likely that the pathological form will block PNKP-mediated TC-SSBR as well. The nervous system encounters a high level of oxidative stress, consuming ~20% of inhaled oxygen. Additionally, postmitotic neurons have a high transcriptional rate, which might further increase the dependency of these cells on TC-SSBR to maintain the integrity of both the nuclear and mt genomes. Therefore, to understand the molecular biology of SSBR and the disease process, our project will have three Specific Aims, to test the hypotheses that: 1. ATXN3-Q72 blocks PNKP-mediated nuclear TC-SSBR; 2. ATXN3-Q72 blocks PNKP-mediated mtTC-SSBR; and 3. Ectopic expression of PNKP will rescue ATXN3-Q72-mediated cellular toxicity. Our long-term goal is to determine the mechanistic basis for the development of Ataxia and to develop new strategies for the prevention or treatment of MJD/SCA3 in the human population.